Differential Gene Expression Between the Porcine Morula and Blastocyst CC Hsu 1 , EC Lin 1 , SC Chen, SC Huang, BH Liu, YH Yu, CC Chen, CC Yang, CY Lien, YH Wang, CW Liu, HJ Mersmann 2 , WTK Cheng and ST Ding Department of Animal Science and Technology, Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Contents The survival and development of pre-implantation embryos are determinant factors affecting the outcome of animal reproduction. It is essential to transfer the expression of the genetic material from maternal sources, that is the ovum to the zygote before implantation to ensure successful development. Differentiation and transformation of blastomeres initiated during the morula and blastocyst stages is an important step of the embryonic development prior to implantation. We col- lected morula and early blastocyst samples from pure-bred Landrace pigs in vivo to study the differential gene expression patterns at these two stages. Total RNA was extracted from individual embryos and two rounds of amplification were employed. Two micrograms of antisense RNA, targets, were prepared and hybridized with each of four custom made oligo microarrays representing 24 000 porcine genes. The analyses of replicate hybridizations showed that among the 24 000 genes, 162 genes were expressed fivefold or greater in the morula compared to early blastocysts and 2126 genes were expressed fivefold or greater in early blastocysts compared to the morula. Of these differentially expressed genes, 1429 genes were functionally annotated with related human Gene Ontology terms. In addition to basic metabolic processes, genes related to signal transduction, transportation and cell differentiation were found in both stages and were up-regulated as embryo development proceeded. Real time polymerase chain reaction was utilized to quantify 12 genes differentially expressed in the 2 embryonic stages and validated the reliability of major evidences shown in microarrays. In conclusion, we have obtained a preliminary landscape of genes differentially expressed during the transition from morula to early blast- ocysts in pigs and showed a generally increased transcriptional activity, perhaps in preparation for implantation. Our results provide an opportunity to study the functions of these genes in relation to the development and survival of pre-implantation porcine embryos. Introduction The first 10–25 days of embryonic development are critical for a successful pregnancy in domestic pigs. A massive embryonic loss usually takes place in this period and results in a decreased litter size (Perry 1954; Perry and Rowlands 1962). There are three major developmental transitions occurring in mammalian pre- implanted embryos, including maternal-to-zygotic tran- sition of the mRNA and protein sources, compaction caused by cell–cell interaction and genetic determina- tion, and the first tissue differentiation of the morula to the blastocyst (Schultz 1993; Schultz et al. 1999; Hamatani et al. 2006). The last event including differ- entiation of blastomeres and formation of the inner cell mass (ICM) and trophectoderm enables the embryo to acquire the ability to implant. There are dramatic changes in gene expression profiles during the formation of blastomeres (Kidder and McLachlin 1985, Kanˇka et al. 1993). Transcripts in oocytes from the maternal source are translated in the embryos as zygotic gene activation and maternal tran- scripts are degraded gradually as embryogenesis pro- ceeds (Flach et al. 1982; Hamatani et al. 2004). Therefore, identification of key genes expressed using functional genomic analysis is important to understand normal early embryonic development. Recently, several studies of the global expression profile of porcine pre-implantation embryos used expressed sequence tag (EST) cDNA libraries (Whit- worth et al. 2004), cDNA microarrays (Whitworth et al. 2005), or GeneFishing polymerase chain reaction (PCR) (Hwang et al. 2005). Because the availability of gene sequences has been improved during the past few years, a set of four oligo-microarrays containing 24 000 genes was constructed from sequences of several embryonic EST libraries. Differential gene expression between the porcine morula and blastocyst were analysed by the microarray and real time PCRs. Materials and Methods Animal care and sample collections Gilts of the Landrace breed (n = 3), whose parents and grandparents were high reproductive performers, were purchased from a local commercial swine breeding farm. They were raised using a standard swine breeder protocol to 10.6 ± 1.2 (mean ± SD) months of age when the embryo samples were taken. The onset of oestrus in these gilts was monitored and then synchro- nized by feeding 20 mg ⁄ day Regument Ò (Intervet, Zu¨rich, Switzerland) for 18 days after observation of the first oestrus cycle. On the nineteenth day, the animals were injected with 1750 IU PMSG im; 80 h later 1000 IU hCG was injected im (China Chemical Pharmaceutical, Taipei, Taiwan) to induce superovula- tion. Gilts were then artificially inseminated 30 h after- wards and a second artificial insemination was done 6 h later to increase the fertilization rate. During the surgical procedure, experimental subjects were first given an anaesthetic (Zoletil containing zolazepam; Virbac Laboratories, Carros, France) at 10 mg ⁄ kg body weight im; and constant anesthesia were maintained by applying a breathing mask to breathe air containing 2–3% halothane (Nicholas Piramal, Mumbai, Mahar- ashtra, India). The embryos were flushed from oviducts 1 These authors contributed equally to this work. 2 Visiting professor at National Taiwan University. Reprod Dom Anim 47, 69–81 (2012); doi: 10.1111/j.1439-0531.2011.01803.x ISSN 0936-6768 Ó 2011 Blackwell Verlag GmbH